The long-term objectives of the project are to better understand the regulation of glial cell function in the mammalian retina and brain by 1) characterizing the potassium channels of retinal astrocytes in situ under normal and pathological conditions and 2) studying the induction of membrane specializations in astrocytes in vitro. The Specific Aims of the project are as follows: 1) Describe the distribution and types of K+ channels in mammalian retinal astrocytes in situ. These experiments will test the hypothesis that K+ channels are concentrate at the endfeet where astrocytes contact retinal blood vessel. 2) Describe the spatial distribution and types of K+ channels in mammalian astrocytes in pure astroglias cultures and in co-cultures with neurons, vascular endothelial cells, and meningeal fibroblasts. These experiments will test the hypothesis that the expression and spatial localization of K+ channels in cultured astrocytes is dependent upon interactions with other cell-types. 3) Determine the types and spatial distribution of K+ channels present on retinal astrocytes under pathological conditions. The hypothesis to be tested is that, under a variety of pathological conditions in which goal reactivity occurs, astrocytic K+ channels will become more numerous and less localized to specific regions of the membrane. 4) Examine the effects of i) alterations in neuronal activity and ii) increases in [K+] on the expression and distribution of K+ channels. Astrocytic K+ channels will be studied electrophysiologically. The spatial distribution of K+ channels will be mapped using hole-cell voltage recordings with patch electrodes to monitor responses of astrocytes to focal ejections of K+. K+ channels will be characterized by their pharmacological sensitivities and by their voltage dependence using cell-attached multi- and single-channel recordings and whole-cell voltage clamp. The types and spatial distribution of K+ channels in astrocytes in situ will be studied in 1) normal isolated rat retinas, 2) retinas damaged by i) a stab wound to the retina, ii) hereditary retinal degeneration (RCD rat), or iii) urethane-induced retinopathy, and 3) retinas in which retinal ganglion cell activity is eliminated by intraocular injection of tetrodotoxin. Inductive effects of other cell- types on astrocytic channel expression will be examine by determining the types and distribution of K+ channels in rat retinal and cortical astrocytes cultured with combinations of neurons, vascular endothelial cells, and meningeal fibroblasts. The expression and distribution of K+ channels will also be studied in cultured rat retinal and cortical astrocytes grown in media with different concentrations of K+. Knowledge of astrocyte membrane properties under normal and pathological conditions is necessary for understanding how these cells function in the normal retina and brain and in clinical conditions such as retinal trauma, retinitis pigmentosa, and epilepsy.